The Interaction Between Aquifex Aeolicus Leucyl-tRNA Synthetase And TRNA～(Leu)

Leucyl-tRNA synthetase (LeuRS) belongs to class I aminiacyl-tRNA synthetase and catalyzes the leucylation of tRNALeu. Aquifex aeolicus αβ-LeuRS is the only known heterodimeric LeuRS, while Escherichia coli LeuRS is a canonical monomeric enzyme. By using the genes encoding A. aeolicus and E. coli LeuRS as PCR templates, the genes encoding the αand βsubunits from A. aeolicus αβ-LeuRS and the equivalent amino-and carboxy-terminal parts of E. coli LeuRS (identified as α'and β') were amplified and recombined using suitable plasmids. These recombinant plasmids were transformed or co-transformed into E. coli to produce five monomeric and five heterodimeric LeuRS mutants. Seven of these were successfully over-expressed in vivo and purified, while three dimeric mutants with the β'part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the αsubunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNALeu occurs at the αsubunit. None of the mixed E. coli-A. aeolicus enzymes were as thermostable as the native αβ-LeuRS. However, the fusion of the two αand βpeptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme. Despite the difference in quaternary structure, the sequence of αβ-LeuRS is close to the Escherichia coli monomeric LeuRS. In the cross recognition of the tRNAs, the A. aeolicus enzyme exhibits a 5-fold decrease of the kcat in the aminoacylation of the E. coli tRNA and a 2-fold decrease of the tRNA affinity, while the E. coli enzyme catalyzes both tRNAs at nearly the same rate. Indeed, subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNALeu occurs at the αsubunit. Furthermore, CP1 domain located in the αsubunit of A. aeolicus LeuRS. Transplantation of the CP1 domain from the E. coli LeuRS into the A. aeolicus enzyme enabled the chimera to enhance the charge of the E. coli tRNALeu counterpart. Conversely, substitution of the CP1 of the E. coli enzyme for that of A. aeolicus increased the charging ability of A. aeolicus tRNALeu slightly. The results showed that CP1 domain of LeuRS was very important for not only the editing reaction, but also the cross-species-specific recognition of tRNALeu. On the other hand, the exchange of CP1 domains in these two LeuRSs impaired their editing abilities. Based on the previous study and the sequence alignment, the similarity and the difference of LeuRS from E. coli and A. aeolicus have been indicated. In order to study the editing function of A. aeolicus LeuRS and compared with that of E. coli LeuRS, mutations of highly conserved T273 and E321 residues in the CP1 domain...